1. Field of Invention
This invention relates, in one aspect, to a biochip reader for reading the wavelengths of fluorescence caused by marking samples, e.g. DNA or protein, with a fluorescent substance and then exciting the marked samples; and, in another aspect, to an electrophoresis system used, for example, in bioengineering; and more particularly, to improvements in such biochip reader and electrophoresis system.
2. Description of the Prior Art
The prior art provides a technique wherein DNA (deoxyribonucleic acid) or protein is marked with a fluorescent substance; then the marked substance is excited by irradiation with laser light, and the resulting wavelengths of fluorescence are read so that the DNA or protein is detected and analyzed. In this technique, a biochip is used with samples of DNA or protein is marked with the fluorescent substance being disposed on the surface thereof in spots or arrays.
The biochip is read by irradiating and scanning laser light laterally, for example, to excite spots of the fluorescent substance arranged in arrays. The emitted fluorescent light is then condensed by an optical fiber, for example, and received by an optical detector through an optical fiber to detect the desired wavelength. When reading of one line or array of spots is completed, the biochip is moved longitudinally to repeat the same process. Then, the process is repeated until the biochip is read entirely.
The conventional biochip reader has the following problems:
(1) The biochip is used to process too many spots, has a large outside dimension, and contains thereon too many arrays of spots.
(2) Fluorescence wavelengths are separated by use of an optical fiber. Thus, it is difficult to separate the wavelengths of polychromatic fluorescent light since any spectra mixture thereof depends on the concentration of each color.
(3) The quantity of measurement deteriorates due to the mixing of fluorescent light with self-emission, background light, or the like. This results in decreased accuracy.
(4) A prolonged period of time is required when switching between optical filters and between optical detectors according to the fluorescent color being detected.
(5) The conventional biochip reader can be speeded up by arranging multiple optical filters and optical detectors and causing the various optical detectors to receive fluorescent light at the same time instead of switching between the filters and the optical detectors. However, this approach increases cost for the added equipment.
(6) Using a scanning confocal microscope with the biochip reader increases the number of system components. This results in increased cost and size, and also the time to perform the requirement measurement is increased.
A biochip, such as a DNA chip, used with the reader has a structure in which several thousand to several ten thousand types of known DNA segments are arranged in arrays on a substrate. If any unknown DNA segment is flowed onto the DNA chip, it is combined with a DNA segment of the same type. Taking advantage of this property of DNA, a known DNA segment, that has formed a combination, is examined by the biochip reader to identify the properties of the unknown DNA, such as DNA arrangement.
FIG. 1 shows an example of hydridizing a biochip, wherein six types of DNA segments DN01-DN06 are arranged in arrays on a substrate SB01 to form a DNA chip. UN01 is an unknown DNA segment and was previously provided a fluorescent mark, as indicated by LM01. When hybridized to the DNA chip, the unknown DNA segment UN01 combines with another DNA segment whose arrangement is complementary. For example, the unknown DNA segment UN01 combines with known DNA segment DN01, as indicated by CB01. Using a biochip reader, excitation light is irradiated at the DNA chip, thus hybridized, in order to detect fluorescent light emitted from the fluorescent mark. Thus, it is possible to determine which of the known DNA segments the unknown DNA segment combined with. For example, in an image resulting from scanning the DNA chip, indicated by SI01, fluorescent light is observed only at a spot where the DNA combination CB01 has been produced. This means fluorescent light is detected only from spot CD01.
FIG. 2 shows an example of a conventional biochip reader, wherein a light source 1 (e.g. a laser) emits excitation light, to a dichroic mirror 2 which reflects light to an objective lens 3 which focuses the light onto a DNA chip 4 which is a biochip onto which a plurality of cells are arranged in an array. The reflected light is transmitted to a filter 5, lens 6 and then to optical detector 7, such as a photo multiplier tube.
The cells CL01-CL03 in which DNA segments, namely, samples, of the same type are arranged on biochip 4.
Light emitted from the light source 1 is reflected by the dichroic mirror 2 as excitation light and condensed onto cells on the DNA chip 4 through the objective lens 3. For example, the excitation light is condensed onto the cell CL02. Fluorescent light produced by the excitation light in cell CL02 becomes parallel light after passing through objective lens 3 and then passes through dichroic mirror 2. Fluorescent light that passed through dichroic mirror 2 then travels through filter 5 and is condensed onto the optical detector 7 by lens 6.
The DNA chip 4 is scanned by a drive means, not shown. For example, the DNA chip 4 is scanned in the direction indicated by arrow MV01 so that the excitation light is irradiated at cells CL01-CL03 on chip 4. Hence, it is possible to identify the arrangement of the unknown DNA segment from the position of a cell where the fluorescence has taken place. That is, fluorescence takes place where the DNA segment to be identified combines with a complementary DNA segment and that combination will be excited to fluoresce.
Unfortunately in most environments, dust may deposit on the DNA chip 4 when mixing foreign matter with a liquid in which the unknown DNA segment is hybridized or when subsequent processes are carried out. If the dust is organic, the excitation light may cause the dust to emit fluorescent light that is more intense than that emitted by a cell. This results in unwanted noise, and deteriorates the S/N ratio.
FIG. 3 is an enlarged view of the cell CL02 of FIG. 2, wherein objective lens 3 and biochip 4 are shown with cell CL02 disposed on the biochip 4. If the DNA chip 4 is contaminated with dust particles, e.g. marked DS01 and DS02, fluorescent light LL11 is produced by the excitation light in addition to fluorescent light emitted from cell CL02. This will cause deterioration of the signal to noise ratio (S/N). For this reason, a confocal optical system has been used as a conventional biochip reader to detect only the fluorescent light produced by the cells by removing fluorescent light produced by the dust. Alternatively, another solution to the dust problem is to hermetically seal the chip 4 and prevent it from being contaminated with dust. However, these measures are not satisfactory because of the problems caused thereby, such as increased cost and insufficiently improved S/N.
In addition, an electrophoresis method has been used to analyze the structure of genes and proteins, such as amino acid, because such method is inexpensive and simple. The methods are often used in the field of bioengineering. The different electrophoresis methods include a disk electrophoresis method using polyacrylamide, an SDS (sodium dedecyl sulfate) polyacrylamide-gel electrophoresis method, an isoelectric point electrophoresis method, a nucleic acid gel electrophoresis method, an electrophoresis method based on the effects of interaction with other molecules, a two dimensional electrophoresis method, and a capillary electrophoresis method.
FIG. 4 shows an exemplary conventional electrophoresis measurement system comprising an electrophoresis unit 10 and a signal processor 20. The electrophoresis unit 10 consists of a lane area 11, a first electrode 12 and a second electrode 13 for applying voltage to the lane area 11, a support plate 14 for supporting the lane area 11 and the first electrode 12 and second electrode 13, a power unit 15 for electrophoresis used to supply voltage to the two electrodes 12 and 13, a light source 16 for emitting light to excite a fluorescent substance, an optical fiber 17 for guiding light emitted by the light source 16, and an optical detector 18 for condensing fluorescent light produced by a fluorescent substance to convert the light to an electric signal after selectively introducing light of a specific wavelength through an optical filter.
The signal processor 20 receives an electric signal from the optical detector 18 to perform appropriate processes, such as converting the electrical signal to digital data or performing preliminary processes, including summing and averaging. The output signal from the processor 20 is supplied to a data processor (not shown) where samples are examined and analyzed.
In the FIG. 4 system, electrophoresis begins when a gel is injected into the lane area 11, samples of DNA segments marked with a fluorescent substance are injected from the gel, and voltage is applied to the first electrode 12 and the second electrode 13 using power unit 15. Molecules contained in the samples gather in each lane of samples as classified by molecular weight, each group of molecules forming a band. Since molecules having lower molecular weight have higher speeds of electrophoresis, they migrate longer distances within the same period of time. These bands are detected by irradiating the gel with laser light, for example, emitted by light source 16, causing marks of the fluorescent substance that concentrate on the bands in the gel to emit fluorescent light, and detecting the fluorescent light with the optical detector 18.
When the gel is irradiated with laser light, the fluorescent substance within part of the gel, which exists along a line L1 shown in FIG. 5, is excited to emit fluorescent light. The fluorescent light is detected at a given position in each lane, as it is searched for in the direction of electrophoresis with the lapse of time. Hence, the fluorescent light is detected when a band B2 of each lane crosses line L1. Thus, it is possible to a signal representing the intensity pattern of fluorescence of a single lane. The data processor which is not shown is designed to analyze each base sequence of the DNA from the pattern signal.
The conventional electrophoresis system has the following problems:
(1) A prolonged time period is required to perform measurement.
(2) The separability of cells is not sufficient. Too many lanes are required in order to separate a variety of DNA segments. Also, information on the correlation among three or more dimensions is not available since the system is limited to two dimensional analysis.
(3) The system requires a large installation space, such as, for example, a lane area as large as 50 cm×50 cm or 5 cm×5 cm.
(4) A two dimensional system is particularly inferior in terms of positional reproducibility. This problem may be solved by applying markers to other lanes and then referencing the added markers. However, applying added markers, disadvantageously, increases lane area needed for analysis.